1. Field of the Invention
The present invention provides compositions comprising blebosomes which express an immunogenic polypeptide specific for a disease, methods of manufacturing the same, methods for detecting antibodies specific for said immunogenic polypeptides and methods for immunizing an animal using said blebosomes.
2. Discussion of the Background
Immunization is a principal feature for improving the health of infants and young children. Despite the availability of a variety of successful vaccines against most of the common childhood illnesses, infectious diseases remain a leading cause of death in children. Significant problems inherent in existing vaccines include the need for repeated immunizations, and the ineffectiveness of the current vaccine delivery systems for a broad spectrum of diseases.
The number of successful approaches to vaccine development is almost as broad as the number of infectious agents. As technology has developed, it has become possible to define at the molecular level the nature of the protective immunogen. In recent years, acellular vaccines have become the method of choice for vaccine development because they can be administered with subunits from a variety of pathogens (i.e. multicomponent vaccines) and they have the potential for reduced numbers of adverse reactions. Subunit vaccines are composed of defined purified protective antigens from pathogenic microorganisms.
Perhaps the best example of success with subunit vaccines is the current vaccine that prevents diseases caused by H. influenzae. A conjugate vaccine composed of H. influenzae polyribosyl ribitol phosphate (PRP) capsular polysaccharide conjugated to an outer membrane protein complex (OMPC) from N. meningitidis has proven to be safe and effective in generating protective immune responses in infants as young as 2 months of age. Covalent coupling of PRP to OMPC results in a conjugate that effectively mediates carrier priming and additionally provides an insoluble particulate antigen containing lipooligo-saccharides (LOS) which have adjuvant activity. This vaccine is widely accepted as safe and effective in reducing the incidence of morbidity and mortality associated with diseases caused by H. influenzae (Stover, C. K. et al., Nature 351:456-460, 1991; Husson, R. N. et al., J. Bacteriol. 172: 519-524, 1990; Jacobs, W. R. et al., Curr. Top. Microbiol. Immunol. 155:153-160, 1990; Jacobs, W. R. et al., Nature 327: 532-535, 1987. All documents cited herein are hereby incorporated by reference).
Although there have been a few stunning successes, a small number of subunit vaccines are currently in use. Perhaps the most daunting reason impeding the use of subunit vaccines is the problem of antigen delivery. For optimal antigen delivery, the antigen needs to be delivered to the antigen presenting cells in its biological context, or the antigen needs to be readily recognized and taken up by phagocytes. Most antigens possess three dimensional structures that are important for parasite-host cell interactions and many of these structures are lost during antigen purification.
One approach taken to circumvent most of the problems associated with subunit vaccine production is the development of live recombinant vaccine vehicles, based on attenuated viruses and bacteria that have been genetically engineered to express protective antigens in vivo (i.e. recombinant forms of vaccinia virus, adenovirus, Salmonella and mycobacterium tuberculosis typhus bonivur var. Bacille-Calmette-Guerin or BCG) (Snapper, S. B. et al., Proc. Natl. Acad. Sci USA 85:6987-6991, 1988; Jackett, P. S. et al., J. Clin. Micro. 26:2313-2318, 1988; Lamb, J. R. et al., Rev. Infect. Dis. 11:S443-S447, 1989; Shinnick, T. M. et al., Infect. Immun. 56:446-451, 1988).
Live vaccines present advantages in that the antigen is expressed in the context of an innately immunogenic form; the live delivery system replicates and persists in the host, restimulating the host immune system and obviating the need for multiple doses; live vector systems eliminate the need to purify the antigen, and are less expensive to produce; and live vectors can be designed to deliver multiple antigens, reducing the number of times an individual must be vaccinated.
Investigators developing Escherichia coli and Salmonella as live vaccine vehicles have developed export vectors utilizing flagella, fimbriae or major outer membrane proteins (OmpA and LamB) as carriers to export protective epitopes to the surface of the bacterial vaccine vehicle (Stover, C. K. et al., Infect. Immun. 58: 1360-1475, 1990; Thole, J. E. R. et al., Infect. Immun. 55:1466-1475, 1988). However, the approach of grafting epitopes into these surfaces is limited, as only small epitopes may be inserted, and the epitopes are presented in a context of a foreign protein that may limit its ability to assume its native conformation.
In order to develop vaccines against pathogens that have been recalcitrant to vaccine development, and/or to overcome the failings of commercially available vaccines due to underutilization, new methods of antigen presentation must be developed which will allow for fewer immunizations, and/or fewer side effects to the vaccine. A new vaccine delivery system is described in this application which is based on Neisseria gonorrhoeae as a host, a bacterium that naturally turns over its outer membrane into easily isolated immunogenic blebs.
N. gonorrhoeae is a human pathogen of mucosal surfaces. N. gonorrhoeae is a Gram-negative bacteria with an undulating outer membrane which appears as a bilayered structure (Reviewed In: The Gonococcus, P. B. Roberts (Ed.). Wiley, New York). The chromosome of the gonococcus contains approximately 2.1.times.10.sup.6 nucleotide pairs. During log phase, the gonococcus forms cell wall blebs which are produced by budding of the outer membrane (Schorr, J. B. et al., Cold Spring Harbor Laboratory Press Vaccines 91:387-392, 1991). These blebs are spherical and are surrounded by a bilayer membrane-like structure. Blebs derived from Neisseria have a liposomal three-dimensional structure and provide immune stimulation (adjuvant activities) to antigens covalently coupled to them (Brandt, M. E. et al., Infect. Immun. 58:983-991, 1989). Protein profiles from gonococcal blebs closely resemble those proteins seen in the outer membrane of the gonococcus (Melchers, F. et al., J. Erp. Med. 142:473-482, 1975). Gonococcal blebs contain on their surface proteins I (Pistor, S. and G. Hobom, Wochenschr. 66:110, 1988), II (Bakker, D. et al., Microb. Path. 8:343-352, 1990), and H8 (Charbit, A. et al., EMBO 5(11):3029-3037, 1986). In addition, they contain gonococcal lipooligosaccharide (LOS).
Much work has been done on characterizing the cell surface antigens of the gonococcus, and many of the genes encoding the protein antigens have been cloned and their DNA sequences determined (Vodkin, M.H. and Williams, J. C., J. Bact. 170:1227-1234, 1988; Young, D. L. et al., Infect. Immun. 54(l):177-183, 1986; Young, D. R. et al., Proc. Natl. Acad. Sci. U.S.A 85:4267-4270, 1998; Newton, S. M. et al., Science 244:70-244, 1989). Studies on biosynthesis and the genetics underlying the biosynthesis of each of the LOS components are far enough along to allow for the successful construction of strains with a defined LOS structure and a stable cell surface. Neisseria mutants that are deficient for LOS and other cell surface proteins have been well described and can be easily produced. Furthermore, gonococcal blebs are easy to isolate and vectors for the genetic manipulations of Neisseria already exist.
Blebosomes are advantageous over liposomes as carriers of antigens. Liposomes are artificially made and proteins of interest are either entrapped in them or chemically conjugated to their surface. The blebosomes of the present invention require no special treatment, and proteins are folded naturally by native enzymes and can be engineered to be expressed inside or outside the bleb.
The present invention provides a vaccine delivery system comprising engineered N. gonorrhoeae wherein, said bacteria expresses and directs any heterologous target antigen to its outer membrane. The outer membrane with the antigenic protein is naturally sloughed off in gonococcal blebs during cell growth. The resulting target antigen-membrane bleb complex or blebosome is easy to isolate and would represent a non-living but immunogenic cell-like particle which can be used to elicit a protective immune response.
Free blebbing, or hyperblebbing strains allow for the necessary production of large amounts of blebosomes for use in a vaccine. In order to commercially produce a vaccine based on gonococcal blebs, it must be possible to produce large quantities of blebosomes. Although all gonococcal strains produce blebs, the yield tends to be low, and prohibitive for use as a vaccine delivery system. A strain that hyperblebs gives the required high yield of blebs for economic production of a vaccine. Such a hyperblebbing strain has not yet been described in the literature. Therefore, there is a need for a strain of N. gonorrhoeae which is able to hyperbleb in order to economically produce quantities of blebosomes containing the target antigens in amounts sufficient for use as a vaccine delivery system.